Project Summary Periodontitis is a highly prevalent disease affecting nearly half of all American adults, and if left untreated leads to bone loss and tissue damage [1]. Multiple microbes are associated with this disease [2, 3] and through chemically-mediated interactions form complex interspecies communities within the periodontal crevice. Due to the complexity of these chemically-mediated interactions, periodontitis remains a difficult disease to treat. Efforts using polymicrobial communities [4, 5] and animal models [5, 6] have explored possible chemical interactions and have greatly advanced our understanding of the chemical interactions occurring during periodontitis. In the Whiteley lab we use a two-species model system composed of Streptococci gordonii (Sg), a representative Gram-positive streptococcal species capable of consuming sugars and producing acids such as L-lactate as well as producing hydrogen peroxide (H2O2), and Aggregatibacter actinomycetemcommitans (Aa), a Gram-negative oral pathogen associated with aggressive periodontitis. Previously, we have shown that when grown in co- culture, Sg cross-feeds Aa its preferred carbon source, L-lactate, while additionally providing the social cue H2O2 thereby enhancing the fitness of Aa [7-9]. By being cross-fed L-lactate, the slow-growing Aa is able to better compete within a polymicrobial environment. Furthermore, H2O2 serves as a cue by stimulating the production of the complement factor ApiA that protects Aa from complement killing [4], and induces the production of the protein Dispersin B that allows Aa to control its spatial localization [9]. In addition to these fitness benefits, we also hypothesize based on previous data that Sg-produced H2O2 also serves as a direct source of O2 for Aa through catalase mediated detoxification [8]. While L-lactate and H2O2 have been shown to provide important metabolic cues for Aa, recent genomic work indicates that there are likely additional chemical interactions occurring between these bacteria during co-infection [8, 10]. Our hypothesis is that Aa displays defined responses to Sg that are critical to establishing precise spatially structured biofilms at the micron scale. The first objective of the project is to test the hypothesis that Aa can use O2 derived from H2O2 detoxification as evidenced by a shift in respiration when Aa is grown in co-culture with Sg, and how H2O2 impacts spatial structure. In the second objective we will use mass spectrometry to develop a comprehensive understanding of the chemical interactions occurring between Aa and Sg. The results from these studies will provide direct insight into the processes underlying the additional benefits Aa receives through H2O2 detoxification. By identifying the unknown chemical interactions between Aa and Sg, we can better understand the complex interspecies interactions involved in periodontitis.